1. Field of the Invention
This invention pertains generally to the field of molecular biology and particularly to techniques of primer dependent polymerase mediated DNA synthesis.
2. Description of Related Art
Relative Quantitative (RQ) RT-PCR.TM. ("reverse transcriptase--polymerase chain reaction") provides a reasonably accurate method of estimating relative levels of expression of individual genes between multiple samples.
To compensate for variations in RNA isolation, initial quantitation errors, and tube to tube variation in RT and PCR.TM. reactions, multiplex RT-PCR.TM. can be performed with an endogenous or exogenously added control amplicon. Multiplex RT-PCR.TM. with internal controls involves amplification using two PCR.TM. primer pairs in the same reaction to co-amplify two targets. One target is the sequence under study, the other is a fragment of an invariant sequence present in all samples. After the amplification reaction, PCR.TM. products are quantified and the yield of the target in each sample is adjusted up or down based on the variance in yield of the internal control amplified in each sample. In practice, this is specifically achieved by calculating the average yield of product per sample for the control product, then calculating the variance of the yield from the average for each individual sample, and finally, adjusting the yield of each experimental target by the inverse of the variance for the control in that sample.
In Competitive RT-PCR.TM., an exogenous target is added as an RNA template to the RT reaction. The level of sample to sample variation in target yield is then adjusted relative to the variation of the reference amplification product. Using an exogenously added standard has the advantage of giving the user absolute control over the amount of template added, with no variation between samples. Thus, the relative abundance of the control target should be adjusted to approximate that of the amplicon under evaluation. This is important because the control must be present in the RT-PCR.TM. at a level similar to the test transcript. If one target is present at a significantly higher concentration than the other, competition for reagents in the PCR.TM. will result in a loss of exponential amplification for the template of lower abundance. Because of competitive interference, the ability to obtain quantitative information will be lost. With this caveat in mind, any clone for which primers are available can be used as an exogenous standard as long as the PCR.TM. product can be differentiated from the experimental target or endogenous source of RNA homologous to the standard. RNA transcripts can be made by in vitro transcription and accurately quantified by including a trace amount of .sup.32 P-UTP in the reaction. A typical in vitro transcription reaction will produce enough RNA for thousands of RT-PCR.TM. reactions. If the first strand synthesis reaction is to be oligo dT-primed, the exogenous control transcript must include a poly (A) tail (as in a cDNA clone, for example). Using an exogenous standard requires considerably more effort compared to using an endogenous standard. Additionally, using such a standard does not control for differences in the quality of the starting RNA in the RT reaction. If there is a difference in the level of intactness of the RNA between otherwise identical samples, the yield of specific RT-PCR.TM. products will reflect this variation although the external standards will still look identical. For this reasons, as well as for simplicity and reproducibility, an endogenous RNA standard is preferred in Semi-Quantitative RT-PCR.TM..
In RQ RT-PCR.TM. product yields are normalized against internal, invariant controls (Horikoshi et al., 1992). The advantages of RQ RT-PCR.TM. over Competitive RT-PCR.TM. is that an exogenous standard is not required and multi-tube titration is not necessary for each sample once reasonable cycling conditions are established. However, the PCR.TM. must be terminated and analyzed while the reaction is in the linear phase for both the target and reference amplicon. This requires pilot experiments to determine cycling parameters designed to avoid plateaus in amplification and the subsequent careful quantitation of products.
Multiplex PCR.TM. is the simultaneous amplification of two or more nucleic acid fragments in the same PCR.TM. reaction. These fragments may be parts of different targets or contained in the same contiguous segment (i.e.: fragments of different genes or fragments of the same gene). Usually, multiple fragments are amplified with multiple PCR.TM. primer pairs. However, if the fragments share common sequences, they may be amplified using a single pair of PCR.TM. primers.
Theoretically, an ideal endogenous standard for RQ RT-PCR.TM. would be a transcript whose expression does not vary during the cell cycle, between cell types, or in response to the experimental treatments that one wishes to examine. Additionally, for an endogenous standard to be valid in multiplex PCR.TM. it is crucial that it be of a similar relative abundance as the target transcript. Unfortunately, such a molecule does not exist and there are serious limitations to the standards currently in use. For example, although .beta. Actin is a frequently used standard (Horikoshi, et al. 1992; Gaudette and Crain, 1991), it's level of expression varies significantly from tissue to tissue. Additionally, co-amplification of an abundant transcript like .beta. Actin or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a reasonable internal control only for transcripts of similar abundance.
Input RNA in RT-PCR.TM. reactions is usually quantified by spectrophotometer. The input RNA in a typical pre-PCR.TM. reverse transcription reaction is total RNA, quantified by absorbency at O.D..sub.260, 80% of which is ribosomal RNA. Furthermore the mRNA component of total cellular RNA can vary from 2% to 5% depending on the tissue. Therefore, even if a transcript was invariant (as expressed as a percentage of mRNA), it's relative abundance would still vary when considered as a percent of total input RNA from different source tissues. Since the majority of the RNA is rRNA, the level of rRNA remains essentially constant from sample to sample. Because 18S and 28S rRNA make up the majority of O.D..sub.260 absorbent material, they should make ideal invariant controls. In fact, 18S and 28S transcripts are frequently used as internal controls in Northern hybridization and RNAse protection assays. However, the abundance of rRNA has been a major limitation to it's utility as a control. For a control to be applicable for RQ RT-PCR.TM., it must be in the linear range of amplification under the same cycling conditions as the target under study (Ferre' et al., 1994). Thus rRNA has not previously proved useful as an RQ RT-PCR.TM. because it is thousands fold too abundant. Several partial solutions to this problem have been proposed, including using less input cDNA for abundant standards in separate PCR.TM. samples (Nicoletti and Sassy-Prigent, 1996) or introducing primers for abundant species at later cycles in the PCR.TM. (Wong et al., 1994). There are severe limitations to both these methods. The first method is not quantitative, and the second is unwieldy and laborious.
While PCR.TM. is the most often utilized method for the amplification of a specific nucleic acid sequence from a complex and often limited amount of starting nucleic acid, several isothermic amplification techniques may also be employed, and would benefit from the development of technologies which would enable the efficiency of a target amplicon to be altered without affecting other targets in a multiplex amplification reaction. Nucleic Acid Sequence Based Amplification (NASBA, Compton, 1991), Self-Sustained Sequence Based Amplification (3SR, Guatelli et al. 1990), and other closely related technologies amplify a specific target RNA sequence from a complex pool of nucleic acid sequences. Although these technologies are slightly different from each other, all rely on the coordinate activities of a retroviral reverse transcriptase and a bacteriophage RNA Polymerase. A hybrid oligonucleotide with a RNA polymerase promoter sequence at it's 5' end and a target specific sequence at it's 3' end is used as a primer by reverse transcriptase for cDNA synthesis. Reverse transcriptase then uses a second gene specific primer to make the cDNA double stranded. Next the phage promoter sequence (now double stranded) incorporated on the cDNA is used by the bacteriophage RNA polymerase for the synthesis of many RNA copies using the cDNA as template. These transcripts become substrates for reverse transcriptase and the cycle begins again. The advantages of isothermic RNA amplification as described is that thermal cycling is not required, the reaction is fast and (unlike in RT-PCR.TM.) contaminating DNA cannot be used as template.
In International Patent WO94/03472, GenProbe describes a method for isothermic amplification of nucleic acid called Transcription Mediated Amplification (TMA). The amplification process described in the claim relies on reverse transcriptase (a primer dependent DNA polymerase) synthesizing cDNA, and RNA polymerase synthesizing RNA. The main amplification event is the RNA synthesis. Within the description of the technique, GenProbe describes the use of blocked or modified primers which are added to the reaction. These function to increase the specificity of the reaction by suppressing spurious side reactions such as primer-dimer formation. Nowhere in the reference is the use of blocked primers mentioned as a method to modulate the efficiency of DNA amplification. Additionally, the reference contains no mention of PCR.TM.. The reference does describe ways to modify oligonucleotides. However, this is knowledge commonly known in the field.